We have recently found that the half-time for flip-flop of lecithin in phospholipid vesicles is greater than eleven days at 37 degrees. This is in contrast with the value of 6.5 hours measured at 30 degrees with spin labels. The electron spin resonance technique has also been used to measure a flip-flop half-time of thirty minutes for the lipid in the red cell membrane. We propose to measure this flip-flop process in the red cell be labelling the outer membrane surface with a 13C-labelled phosphatidyl choline using an exchange process mediated by the phospholipid exchange protein (PLEP). The disappearance of the labelled lipid from the outer membrane surface, and thus flip-flop, will be monitored by measuring the 13C-nuclear magnetic resonance (NMR) spectrum in the presence of a chemical shift reagent. Initially, we will verify this technique by measurement of flip-flop in lipid vesicles, prepared with a 13C-labelled lecithin present solely in the outer surface of the bilayer. These experiments with the model system will enable us to develop the methodology required to produce sufficient quantities of asymmetrically labelled membranes necessary for the NMR measurement. This will in part involve several preparations of the PLEP. If application of these measurements to the red cell reveals a movement of lipid across the membrane, similar experiments will then be conducted with sealed inside-out and right-side-out vesicles derived from the erythrocyte membrane, to determine the flip-flop rate in both directions across the membrane. If no flip-flop is observed in the intact red cell, we will then attempt to measure the same process in the influenza virus to determine whether it occurs in other plasma membranes. BIBLIOGRAPHIC REFERENCES: Rothman, J.E., Tsai, D.K., Dawidowicz, E.A. and Lenard J. 1976. Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus. Biochemistry 15, 2361. Dawidowicz, E.A. and Rothman, J.E. 1976. Fusion and protein-mediated phospholipid exchange studied with single bilayer phosphatidylcholine vesicles of different density. Biochim. Biophys. Acta 455, 621.